Nucleotide sequences coding for the thrE gene and process for the enzymatic production of L-threonine using coryneform bacteria

ABSTRACT

The invention relates to preferably recombinant DNA derived from  Corynebacterium  and replicable in coryneform microorganisms, which contains at least one nucleotide sequence that codes for the thrE gene, and a process for the production of L-threonine, which is characterised in that the following steps are carried out:
     a) Fermentation of microorganisms in which at least the thrE gene is amplified (overexpressed), optionally in combination with further genes,   b) Enrichment of the L-threonine in the medium or in the cells of the microorganisms, and   c) Isolation of the L-threonine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/431,099filed on Nov. 1, 1999, now U.S. Pat. No. 6,410,705.

The present invention relates to nucleotide sequences coding for thethrE gene and a process for the enzymatic production of L-threonineusing coryneform bacteria, in which the thrE gene is amplified.

PRIOR ART

L-threonine is used in animal nutrition, in human medicine and in thepharmaceutical industry.

It is known that L-threonine can be produced by fermentation of strainsof coryneform bacteria, in particular Corynebacterium glutamicum. Onaccount of the great importance of L-threonine, attempts are constantlybeing made to improve the production processes. Production improvementsmay relate to fermentation technology measures such as for examplestirring and provision of oxygen, or the composition of the nutrientmedium such as for example the sugar concentration during fermentation,or the working-up to the product form by for example ion exchangechromatography, or the intrinsic production properties of themicroorganism itself.

Methods employing mutagenesis, selection and choice of mutants are usedto improve the production properties of these microorganisms. In thisway strains are obtained that are resistant to antimetabolites such asfor example the threonine analogon α-amino-β-hydroxyvaleric acid (AHV)or are auxotrophic for regulatory significant amino acids and produceL-threonine.

For some years now recombinant DNA technology methods have also beenused for the strain improvement of L-threonine producing strains ofCorynebacterium, by amplifying individual threonine biosynthesis genesand investigating the action on L-threonine production.

OBJECT OF THE INVENTION

The inventors have aimed to provide new measures for the improvedenzymatic production of L-threonine.

DESCRIPTION OF THE INVENTION

L-threonine is used in animal nutrition, in human medicine and in thepharmaceutical industry. There is therefore a general interest inproviding new improved processes for producing L-threonine.

The object of the invention is a preferably recombinant DNA derived fromCorynebacterium and replicable in coryneform microorganisms, whichcontains at least the nucleotide sequence coding for the thrE gene,represented in the sequences SEQ-ID-No. 1 and SEQ-ID-No. 3.

The object of the invention is also a replicable DNA according to claim1 with:

-   (i) the nucleotide sequences shown in SEQ-ID-No. 1 or SEQ-ID-No. 3,    that code for the thrE gene, or-   (ii) at least one sequence that corresponds to the sequences (i)    within the degeneration region of the genetic code, or-   (iii) at least one sequence that hybridises with the sequence    complementary to the sequences (i) or (ii), and/or optionally-   (iv) functionally neutral sense mutations in (i).

The object of the invention are also coryneform microorganisms, inparticular of the genus Corynebacterium, transformed by the introductionof the aforementioned replicable DNA.

The invention finally relates to a process for the enzymatic productionof L-threonine using coryneform bacteria, which in particular alreadyproduce L-threonine and in which the nucleotide sequence, (s) coding forthe thrE gene is/are amplified, in particular overexpressed.

The term “amplification” describes in this connection the enhancement ofthe intracellular activity of one or more enzymes in a microorganismthat are coded by the corresponding DNA, by for example increasing thecopy number of the gene or genes or using a strong promoter or a genethat codes for a corresponding enzyme having a high activity, and ifnecessary using a combination of these measures.

The microorganisms that are the object of the present invention canproduce L-threonine from glucose, sucrose, lactose, fructose, maltose,molasses, starch, cellulose or from glycerol and ethanol. Themicroorganisms may be representatives of coryneform bacteria, inparticular of the genus Corynebacterium. In the genus Corynebacteriumthe species Corynebacterium glutamicum should in particular bementioned, which is known to those in the specialist field for itsability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum, are in particular the known wildtype strains

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum ATCC13870    -   Corynebacterium melassecola ATCC17965    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020        and L-threonine-producing mutants or strains obtained thereform,        for example    -   Corynebacterium glutamicum ATCC21649    -   Brevibacterium flavum BB69    -   Brevibacterium flavum DSM5399    -   Brevibacterium lactofermentum FERM-BP 269    -   Brevibacterium lactofermentum TBB-10

The inventors have successfully managed to isolate the thrE gene ofCorynebacterium glutamicum. In order to isolate the thrE gene a mutantof C. glutamicum defective in the thrE gene is first of all produced. Tothis end a suitable starting strain such as for example ATCC14752 orATCC13032 is subjected to a mutagenesis process.

Conventional mutagenesis processes include treatment with chemicals, forexample N-methyl-N-nitro-N-nitrosoguanidine, or UV irradiation. Suchprocesses for initiating mutation are generally known and may beconsulted in, inter alia, Miller (A Short Course in Bacterial Genetics,A Laboratory Manual and Handbook for Escherichia coli and RelatedBacteria (Cold Spring Harbor Laboratory Press, 1992)) or in the handbook“Manual of Methods for General Bacteriology” The American Society forBacteriology (Washington D.C., USA, 1981).

Another mutagenesis process is the method of transposon mutagenesis inwhich the property of a transposon is utilised to “jump” in DNAsequences and thereby interfere with or switch off the function of therelevant gene. Transposons of coryneform bacteria are known in thespecialist field. For example, the erythromycin resistance transposonTn5432 (Tauch et al., Plasmid (1995) 33: 168-179) and thechloramphenicol resistance transposon Tn5546 have been isolated fromCorynebacterium xerosis strain M82B.

Another transposon is the transposon Tn5531 described by Ankri et al.(Journal of Bacteriology (1996) 178: 4412-4419) and that was used forexample in the course of the present invention. The transposon Tn5531contains the aph3 kanamycin resistance gene and can be delivered forexample in the form of the plasmid vector pCGL0040, which is shown inFIG. 1. The nucleotide sequence of the transposon Tn5531 is freelyavailable under the accession number U53587 from the National Center forBiotechnology Information (NCBI, Bethesda, Md., USA).

After mutagenesis, preferably transposon mutagenesis, has been carriedout a search is made for a mutant defective in the thrE gene. A mutantdefectve in the thrE gene is recognised by the fact that it exhibitsgood growth on minimal agar, but poor growth on minimal agar that hasbeen supplemented with threonine-containing oligopeptides, for examplethe tripeptide threonyl-threonyl-threonine.

An example of such a mutant is the strain ATCC14752ΔilvAthrE::Tn5531.

A strain produced in the described manner may be used to isolate andclone the thrE gene.

To this end a gene bank of the bacterium that is of interest may beestablished. The establishment of gene banks is recorded in generallyknown textbooks and manuals. There may be mentioned by way of examplethe textbook by Winnacker: Gene und Klone, eine Einfuhrung in dieGentechnologie (Gene and Clones, An Introduction to Gene Technology)(Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook etal.: Molecular Cloning, A Laboratory Manual (Cold Spring HarborLaboratory Press, 1989). A very well-known gene bank is that of the E.coli K-12 strain W3110, which has been established by Kohara et al.(Cell 50, 495-508 (1987)) in λ-vectors. Bathe et al. (Molecular andGeneral Genetics, 252:255-265, 1996) describes a gene bank of C.glutamicum ATCC13032, which has been established in the E. coli K-12strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575)with the aid of the cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA, 84:2160-2164). Forthe present invention those vectors are suitable that replicate incoryneform bacteria, preferably Corynebacterium glutamicum. Such vectorsare known from the prior art; the plasmid vector pZ1 may be mentioned asan example, which is described by Menkel et al. (Applied andEnvironmental Microbiology (1989) 64: 549-554). The gene bank obtainedin the described way is then converted by means of transformation orelectroporation into the indicator strain defective in the thrE gene andthose transformants are sought that have the ability to grow on minimalagar in the presence of threonine-containing oligopeptides. The clonedDNA fragment may then be subjected to a sequence analysis.

When using a mutant of a coryneform bacterium produced by Tn5531mutagenesis, for example the strain ATCC14752ΔilvAthrE::Tn5531, thethrE::Tn5531 allele may be cloned directly using the kanamycinresistance gene aph3 contained in the latter and isolated. For thispurpose known cloning vectors are used, such as for example pUC18(Norrander et al., Gene (1983) 26: 101-106 and Yanisch-Perron et al.,Gene (1985) 33: 103-119). Particularly suitable as cloning hosts arethose E. coli strains that are both restriction-defective andrecombinant-defective. An example is the strain DH5αmcr, which has beendescribed by Grant et al. (Proceedings of the National Academy ofSciences USA, 87 (1990) 4645-4649). The selection for transformants iscarried out in the presence of kanamycin. The plasmid DNA of theresultant transformants is then sequenced. For this purpose the dideoxychain termination method described by Sanger et al. may be used(Proceedings of the National Academy of Sciences of the United States ofAmerica USA (1977) 74: 5463-5467). The thrE gene sequences upstream anddownstream of the Tn5531 insertion site are thereby obtained. Theresultant nucleotide sequences are then analysed and assembled withcommercially available sequence analysis programs, for example with theprogram package Lasergene (Biocomputing Software for Windows, DNASTAR,Madison, USA) or the program package HUSAR (Release 4.0, EMBL,Heidelberg, Germany).

In this way the new DNA sequence of C. glutamicum coding for the thrEgene was obtained, which as SEQ ID NO 1 is a constituent part of thepresent invention. The amino acid sequence of the corresponding proteinhas also been derived from the present DNA sequence using theaforedescribed methods. The resulting amino acid sequence of the thrEgene product is represented in SEQ ID NO 2.

Coding DNA sequences that are produced from SEQ ID NO 1 by thedegenerability of the genetic code are likewise a constituent part ofthe invention. In the same way, DNA sequences that hybridise with SEQ IDNO 1 or parts of SEQ ID NO 1 are a constituent part of the invention.Furthermore, conservative amino acid exchanges, for example the exchangeof glycine by alanine or of aspartic acid by glutamic acid in proteinsare known in the specialist field as sense mutations, which do not causeany fundamental change in the activity of the protein, i.e. arefunctionally neutral. It is furthermore known that changes at the N-and/or the C-terminus of a protein do not substantially affect itsfunction or may even stabilise it. The person skilled in the art mayfind details of this in, inter alia, Ben-Bassat et al. (Journal ofBacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251(1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), inHochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in knowntextbooks on genetics and molecular biology. Amino acid sequences thatare produced in a corresponding manner from SEQ ID NO 2 are likewise aconstituent part of the invention.

Suitable primers can be synthesised using the nucleotide sequence shownin SEQ ID NO. 1 and these are then used to amplify by means of thepolymerase chain reaction (PCR) thrE genes of various coryneformbacteria and strains. The person skilled in the art may find details ofthis in for example the manual by Gait: Oligonucleotide synthesis: apractical approach (IRL Press, Oxford, UK, 1984) and in Newton andGraham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).Alternatively, the nucleotide sequence shown in SEQ ID NO. 1 or partsthereof may be used as a probe to search for thrE genes in gene banks ofin particular coryneform bacteria. The person skilled in the art canfind details of this in for example the manual “The DIG System UsersGuide for Filter Hybridization” published by Boehringer Mannheim GmbH(Mannheim, Germany, 1993) and in Liebl et al. (International Journal ofSystematic Bacteriology (1991) 41: 255-260). The thrE gene-containingDNA fragments amplified in this way are then cloned and sequenced.

The DNA sequence of the thrE gene of the strain ATCC13032 illustrated inSEQ ID NO. 3 was obtained in this way, and is likewise a constituentpart of the present invention. The resultant amino acid sequence isshown in SEQ ID NO. 4.

The invention also provides a process for isolating the thrE gene,characterised in that mutants, preferably of coryneform bacteria,defective in the thrE gene are obtained as indicator strains that do notgrow or grow only weakly on a nutrient medium containing athreonine-containing oligopeptide, and

-   -   a) the thrE gene is identified and isolated after establishing a        gene bank, or    -   b) in the case of transposon mutagenesis is selected for the        transposon preferably exhibiting resistance to antibiotics, and        the thrE gene is thereby obtained.

The inventors discovered from this that coryneform bacteria afterover-expression of the thrE gene produce L-threonine in an improvedmanner

In order to achieve an over-expression, the copy number of thecorresponding genes can be increased, or the promoter and regulationregion or the ribosome binding site located upstream of the structuregene can be mutated. Expression cassettes that are incorporated upstreamof the structure gene work in the same way. It is in addition possibleto enhance the expression during the course of the enzymatic L-threonineproduction by inducible promoters. The expression is also improved bymeasures aimed at lengthening the lifetime of the m-RNA. The enzymaticactivity can also be increased by preventing the decomposition of theenzyme protein. The genes or gene constructs may be present either inplasmids with different copy numbers or may be integrated and amplifiedin the chromosome. Alternatively, an over-expression of the relevantgenes can also be achieved by changing the composition of the culturemedia and cultivation conditions.

A person skilled in the art can find details of this in, inter alia,Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al.(Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6,428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), inEuropeän Patent Specification EPS 0 472 869, in U.S. Pat. No. 4,601,893,in Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991), in Reinscheidet al. (Applied and Environmental Microbiology 60, 126-132 (1994)), inLaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), inPatent application WO 96/15246, in Malumbres et al. (Gene 134, 15-24(1993)), in Japanese laid-open specification JP-A-10-229891, in Jensenand Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), inMakrides (Microbiological Reviews 60:512-538 (1996)) and in knowntextbooks on genetics and molecular biology.

An example of a plasmid by means of which the thrE gene can beoverexpressed is pZ1thrE (FIG. 2), which is contained in the strainDM368-2 pZ1thrE. Plasmid pZ1thrE is a C. glutamicum—E. coli shuttlevector based on plasmid pZ1, which is described by Menkel et al.(Applied and Environmental Microbiology (1989) 64: 549-554). Otherplasmid vectors replicable in C. glutamicum, such as for example pEKEx1(Eikmanns et al., Gene 102:93-98 (1991)) or pZ8-1 (EP-B-0 375 889) canbe used in the same way.

In addition, it may be advantageous for the production of L-threonine toover-express, in addition to the new thrE gene, one or more enzymes ofthe known threonine biosynthesis pathway or enzymes of the anapleroticmetabolism or enzymes of the citric acid cycle. The following may forexample be simultaneously overexpressed:

-   -   the hom gene coding for homoserine dehydrogenase (Peoples et        al., Molecular Microbiology 2, 63-72 (1988)) or the hom^(dr)        allele coding for a feedback-resistant homoserine dehydrogenase        (Archer et al. Gene 107, 53-59, (1991)), or    -   the pyc gene (DE-A-19 831 609) coding for pyruvate carboxylase,        or    -   the mqo gene coding for malate:quinone oxidoreductase (Molenaar        et al., European Journal of Biochemistry 254, 395-403 (1998)).

For the production of L-threonine it may furthermore be advantageous, inaddition to the over-expression of the thrE gene, to exclude undesirablesecondary reactions, such as for example the threonine-dehydrogenasereaction (Nakayama: “Breeding of Amino Acid Producing Micro-organisms”,in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek(eds.), Academic Press, London, UK, 1982 and Bell and Turner,Biochemical Journal 156, 449-458 (1976)).

The microorganisms produced according to the invention may be cultivatedcontinuously or batchwise in a batch process (batch cultivation) or in afed batch (feed process) or repeated fed batch process (repetitive feedprocess) for the purposes of producing L-threonine. A summary of knowncultivation methods is given in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (GustavFischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,Brunswick/Wiesbaden, 1994)).

The culture medium to be used must satisfy in an appropriate manner therequirements of the respective strains. Descriptions of culture mediafor various microorganisms are given in “Manual of Methods for GeneralBacteriology” The American Society for Bacteriology (Washington D.C.,USA, 1981). Sources of carbon that may be used include sugars andcarbohydrates, for example glucose, sucrose, lactose, fructose, maltose,molasses, starch and cellulose, oils and fats such as soybean oil,sunflower oil, groundnut oil and coconut oil, fatty acids such aspalmitic acid, stearic acid and linoleic acid, alcohols such as glyceroland ethanol, and organic acids such as acetic acid. These substances maybe used individually or as a mixture. Sources of nitrogen that may beused include organic compounds containing nitrogen such as peptones,yeast extract, meat extract, malt extract, corn steep liquor, soybeanmeal and urea, or inorganic compounds such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.The sources of nitrogen may be used individually or as a mixture.Sources of phosphorus that may be used include phosphoric acid,potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or thecorresponding sodium salts. The culture medium must furthermore containsalts of metals such as for example magnesium sulfate or iron sulfate,which are necessary for growth. Finally, essential growth substancessuch as amino acids and vitamins may be used in addition to theaforementioned substances. Moreover, suitable precursors may be added tothe culture medium. The aforementioned substances may be added to theculture in the form of a single batch or in an appropriate manner duringthe cultivation.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammoniaor ammonia water, or acidic compounds such as phosphoric acid orsulfuric acid may be added in an appropriate manner in order to controlthe pH of the culture. Anti-foaming agents such as for example fattyacid polyglycol esters may be used to control foam formation. Suitableselectively acting substances, for example antibiotics, may be added tothe medium in order to maintain the stability of plasmids. Oxygen oroxygen-containing gas mixtures, for example air, may be fed into theculture to maintain aerobic conditions. The temperature of the cultureis normally 20° C. to 45° C., and preferably 25° C. to 40° C.Cultivation is continued until a maximum amount of L-threonine has beenformed. This target is normally achieved within 10 to 160 hours.

The analysis of L-threonine can be carried out by anion exchangechromatography followed by ninhydrin derivatisation as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190), or can becarried out by reversed phase HPLC as described by Lindroth et al.(Analytical Chemistry (1979) 51: 1167-1174).

The following microorganisms have been registered according to theBudapest Treaty at the German Collection for Microorganisms and CellCultures (DSMZ, Brunswick, Germany):

-   -   Brevibacterium flavum strain DM368-2 pZ1thrE as DSM 12840    -   Escherichia coli strain GM2929pCGL0040 as DSM 12839

EXAMPLES

The present invention is described in more details hereinafter with theaid of examples of implementation.

The isolation of plasmid DNA from Escherichia coli as well as alltechniques for restriction, Klenow and alkaline phosphatase treatmentwere carried out according to Sambrook et al. (Molecular cloning. Alaboratory manual (1989) Cold Spring Harbour Laboratory Press). Unlessotherwise specified, the transformation of Escherichia coli was carriedout according to Chung et al. (Proceedings of the National Academy ofSciences of the United States of America USA (1989) 86: 2172-2175).

Example 1

Cloning and Sequencing of the thrE Gene of Corynebacterium glutamicumATCC14752

1. Transposon Mutagenesis and Choice of Mutants

The strain Corynebacterium glutamicum ATCC14752ΔilvA was subjected tomutagenesis with the transposon Tn5531, whose sequence is filed underAccession No. U53587 in the Nucleotide Databank of the National Centerfor Biotechnology Information (Bethesda, USA). The incorporation of adeletion into the ilvA gene of Corynebacterium glutamicum ATCC14752 wascarried out with the gene exchange system described by Schäfer et al.(Gene (1994) 145: 69-73). To this end, the inactivation vectorpK19mobsacBΔilvA (Applied and Environmental Microbiology (1999) 65:1973-1979) constructed by Sahm et al. was used for the deletion. Themethylase-defective Escherichia coli strain SCS110 (Jerpseth and Kretz,STRATEGIES in molecular biology 6, 22, (1993)) from Stratagene(Heidelberg, Germany) was first of all transformed with 200 ng of thevector pK19mobsacBΔilvA. Transformants were identified by means of theirkanamycin resistence on 50 μg/mL kanamycin-containing LB-agar plates.The plasmid pK19mobsacBΔilvA was prepared from one of the transformants.This inactivation plasmid was then introduced into the strainCorynebacterium glutamicum ATCC14752 by means of electroporation (Hayneset al., FEMS Microbiology Letters (1989) 61: 329-334). Clones in whichthe inactivation vector was present integrated in the genome wereidentified by means of their kanamycin resistance on 15 μg/mLkanamycin-containing LBHIS-agar plates (Liebl et al., FEMS MicrobiologyLetters (1989) 65: 299-304). In order to select for the excision of thevector, kanamycin-resistant clones were plated out on sucrose-containingLBG-Medium (LB-Medium with 15 g/L agar, 2% glucose and 10% sucrose).Colonies were obtained in this way which had lost the vector through asecond recombination event (Jäger et al.; Journal of Bacteriology (1992)174: 5462-5465). By hetero-inoculation on minimal medium plates(CGXII-Medium with 15 g/L agar (Keilhauer et al., Journal ofBacteriology (1993) 175: 5595-5603)) with and without 300 mg/L ofL-isoleucine, and with and without 50 μg/mL of kanamycin, six cloneswere isolated that by excision of the vector were kanamycin sensitiveand isoleucine auxotrophic and in which only the incomplete ilvA-Gen(ΔilvA allele) was present in the genome. One of these clones wasdesignated strain ATCC14752ΔilvA and used for the transposonmutagenesis.

The plasmid pCGL0040, which contains the assembled transposon Tn5531(Ankri et al., Journal of Bacteriology (1996) 178: 4412-4419) wasisolated from the methylase-defective E. coli strain GM2929pCGL0040 (E.coli GM2929: Palmer et al., Gene (1994) 143: 1-12). The strainCorynebacterium glutamicum ATCC14752ΔilvA was transformed with theplasmid pCGL0040 by means of electroporation (Haynes et al., FEMSMicrobiology Letters (1989) 61: 329-334). Clones in which the transposonTn5531 was integrated into the genome were identified by means of theirkanamycin resistance on 15 μg/mL kanamycin-containing LBHIS-agar plates(Liebl et al., FEMS Microbiology Letters (1989) 65: 299-304). In thisway 2000 clones were obtained which were checked for retarded growth inthe presence of threonyl-threonyl-threonine. For this purpose all cloneswere transferred individually to CGXII minimal medium agar plates withand without 2 mM threonyl-threonyl-threonine. The medium was identicalto the medium CGXII described by Keilhauer et al. (Journal ofBacteriology (1993) 175: 5593-5603), but in addition contained 25 μg/mLof kanamycin, 300 mg/L of L-isoleucine and 15 g/L of agar. Thecomposition of the medium described by Keilhauer et al. is shown inTable 1.

TABLE 1 Composition of the Medium CGXII Component Concentration(NH₄)₂SO₄ 20 g/L Urea 5 g/L KH₂PO₄ 1 g/L K₂HPO₄ 1 g/L MgSO₄ × 7 H₂O 0.25g/L 3-morpholinopropanesulfonic acid 42 g/L CaCl₂ 10 mg/L FeSO₄ × 7 H₂O10 mg/L MnSO₄ × H₂O 10 mg/L ZnSO₄ × 7H₂O 1 mg/L CuSO₄ 0.2 mg/L NiCl₂ × 6H₂O 0.02 mg/L Biotin 0.2 mg/L Glucose 40 g/L Protocatechuic Acid 30 mg/L

The agar plates were incubated at 30° C. and the growth was investigatedafter 12, 18 and 24 hours. A transposon mutant was obtained that grew ina comparable manner to the initial strain Corynebacterium glutamicumATCC14752ΔilvA without threonyl-threonyl-threonine, but which in thepresence of 2 mM threonyl-threonyl-threonine exhibited retarded growth.This was designated ATCC14752ΔilvAthrE::Tn5531.

2. Cloning and Sequencing of the Insertion Site of Tn5531 inATCC14752ΔilvAthrE::Tn5531.

In order to clone the insertion site located upstream of the transposonTn5531 in the mutant described in Example 1.1, the chromosomal DNA ofthis mutant strain was first of all isolated as described by Schwarzeret al. (Bio/Technology (1990) 9: 84-87) and 400 ng of the latter was cutwith the restriction endonuclease EcoRI. The complete restriction insertwas ligated with the vector pUC18 likewise linearised with EcoRI(Norander et al., Gene (1983) 26: 101-106) from Roche Diagnostics(Mannheim, Germany). The E. coli strain DH5αmcr (Grant et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica USA (1990) 87: 4645-4649) was transformed with the completeligation insert by means of electroporation (Dower et al., Nucleic AcidResearch (1988) 16: 6127-6145). Transformants in which the insertionsites of the transposon Tn5531 were present cloned on the vector pUC18were identified by means of their carbenicillin resistance and kanamycinresistance on LB-agar plates containing 50 μg/mL of carbenicillin and 25μg/mL of kanamycin. The plasmids were prepared from three of thetransformants and the sizes of the cloned inserts were determined byrestriction analysis. The nucleotide sequence of the insertion site onone of the plasmids was determined with a ca. 5.7 kb large insert by thedideoxy chain termination method of Sanger et al. (Proceedings of theNational Academy of Sciences of the United States of America USA (1977)74: 5463-5467). For this purpose 2.2 kb of the insert were sequencedstarting from the following oligonucleotide primer: 5′-CGG GTC TAC ACCGCT AGC CCA GG-3′. (SEQ ID NO:5)

In order to identify the insertion site located downstream of thetransposon, the chromosomal DNA of the mutant was cut with therestriction endonuclease XbaI and ligated in the vector pUC18 linearisedwith XbaI. The further cloning was carried out as described above. Thenucleotide sequence of the insertion site on one of the plasmids wasdetermined with a ca. 8.5 kb large insert by the dideoxy chaintermination method of Sanger et al. (Proceedings of the National Academyof Sciences of the United States of America USA (1977) 74: 5463-5467).For this purpose 0.65 kb of the insert was sequenced starting from thefollowing oligonucleotide primer: 5′-CGG TGC CTT ATC CAT TCA GG-3′. (SEQID NO:6)

The obtained nucleotide sequences were analysed and assembled with theprogram package Lasergene (Biocomputing Software for Windows, DNASTAR,Madison, USA). This nucleotide sequence is reproduced as SEQ ID NO 1.The analysis identified an open reading frame 1467 bp long. Thecorresponding gene was designated the thrE gene. The associated geneproduct comprises 489 amino acids and is reproduced as SEQ ID NO 2.

Example 2

Cloning and Sequencing of the Gene thrE from Corynebacterium glutamicumATCC13032

The gene thrE was cloned in the E. coli cloning vector pUC18 (Norranderet al., Gene (1983) 26: 101-106, Roche Diagnostics, Mannheim, Germany).The cloning was carried out in two stages. The gene from Corynebacteriumglutamicum ATCC13032 was first of all amplified by a polymerase chainreaction (PCR) by means of the following oligonucleotide primer derivedfrom SEQ ID NO 1

ThrE-forward: 5′-CCC CTT TGA CCT GGT GTT ATT G-3′ (SEQ ID NO:7)thrE-reverse: 5′-CGG CTG CGG TTT CCT CTT-3′ (SEQ ID NO:8)

The PCR reaction was carried out in 30 cycles in the presence of 200 μMof deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) and, for each,1 μM of the corresponding oligonucleotide, 100 ng of chromosomal DNAfrom Corynebacterium glutamicum ATCC13032, {fraction (1/10)} volumes of10-fold reaction buffer and 2.6 units of a heat-stable Taq-/Pwo-DNApolymerase mixture (Expand High Fidelity PCR System from RocheDiagnostics, Mannheim, Germany) in a Thermocycler (PTC-100, MJ Research,Inc., Watertown, USA) under the following conditions: 94° C. for 30seconds, 58° C. for 30 seconds and 72° C. for 2 minutes.

The amplified, about 1.9 kb large fragment was then ligated using theSureClone Ligation Kit (Amersham Pharmacia Biotech, Uppsala, Sweden)according to the manufacturer's instructions, into the SmaI cleavagesite of the vector pUC18. The E. coli strain DH5αmcr (Grant et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica USA (1990) 87: 4645-4649) was transformed with the wholeligation insert. Transformants were identified on the basis of theircarbenicillin resistance on 50 μg/mL carbenicillin-containing LB agarplates. The plasmids were prepared from 8 of the tranformants and testedby restriction analysis for the presence of the 1.9 kb PCR fragment asinsert. The resultant recombinant plasmid is designated hereinafter aspUC18thrE.

The nucleotide sequence of the 1.9 kb PCR fragment in plasmid pUC18thrEwas determined by the dideoxy chain termination method of Sanger et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica USA (1977) 74: 5463-5467). For this purpose the complete insertof pUC18thrE was sequenced with the aid of the following primers fromRoche Diagnostics (Mannheim, Germany).

Universal primer: 5′-GTA AAA CGA CGG CCA GT-3′ (SEQ ID NO:9)

Reverse primer: 5′-GGA AAC AGC TAT GAC CAT G-3′ (SEQ ID NO:10)

The nucleotide sequence is reproduced as SEQ ID NO 3. The containednucleotide sequence was analysed using the program package Lasergene(Biocomputing Software for Windows, DNASTAR, Madison, USA). The analysisidentified an open reading frame 1467 bp long, which was designated thethrE gene. This codes for a polypeptide of 489 amino acids, which isreproduced as SEQ ID NO 4.

Example 3

Expression of the Gene thrE in Corynebacterium glutamicum

The gene thrE from Corynebacterium glutamicum ATCC13032 described inExample 2 was cloned for expression in the vector pZ1 (Menkel et al.,Applied and Environmental Microbiology (1989) 64: 549-554). For thispurpose a 1881 bp large DNA fragment containing the gene thrE wasexcised from the plasmid pUC18thrE using the restriction enzymes SacIand XbaI. The 5′- and 3′-ends of this fragment were treated with Klenowenzyme. The resulting DNA fragment was ligated in the vector pZ1previously linearised and dephosphorylated with ScaI. The E. coli strainDH5αmcr (Grant et al., Proceedings of the National Academy of Sciencesof the United States of America USA (1990) 87: 4645-4649) wastransformed with the whole ligation insert. Tranformants were identifiedon the basis of their kanamycin resistance on 50 μg/mLkanamycin-containing LB agar plates. The plasmids were prepared from twotransformants and checked by restriction analysis for the presence ofthe 1881 bp ScaI/XbaI fragment as insert. The recombinant plasmidproduced in this way was designated pZ1thrE (FIG. 2).

The plasmids pZ1 and pZ1thrE were incorporated by means ofelectroporation (Haynes et al., FEMS Microbiology Letters (1989) 61:329-334) into the threonine-forming strain Brevibacterium flavumDM368-2. The strain DM368-2 is described in EP-B-0 358940 and is filedas DSM5399. Transformants were identified on the basis of theirkanamycin resistance on 15 μg/mL kanamycin-containing LBHIS-agar plates(Liebl et al., FEMS Microbiology Letters (1989) 65: 299-304). Thestrains Brevibacterium flavum DM368-2 pZ1 and DM368-2 pZ1thrE wereobtained in this way.

Example 5

Preparation of L-threonine with Brevibacterium flavum

In order to investigate their threonine formation the strains B. flavumDM368-2 pZ1 and DM368-2 pZ1thrE were precultivated in 100 mL of brainheart infusion medium together with 50 μg of kanamycin/mL (DifcoLaboratories, Detroit, USA) for 14 hours at 30° C. The cells were thenwashed once with 0.9% (w/v) of sodium chloride solution and 60 mLportions of CgXII medium were inoculated with this suspension so thatthe OD₆₀₀ (optical density at 600 nm) was 0.5. The medium was identicalto the medium described by Keilhauer et al. (Journal of Bacteriology(1993) 175: 5593-5603), but contained in addition 50 μg of kanamycin permL. Both strains were cultivated at 30° C. over a period of 72 hours.Samples were taken after 0, 24, 48 and 72 hours and the cells werequickly centrifuged off (5 minutes at 13000 RPM in a Biofuge pico fromHeraeus, Osterode, Germany).

The quantitative determination of the extracellular amino acidconcentrations from the culture supernatant was carried out by means ofreversed phase HPLC (Lindroth et al., Analytical chemistry (1979) 51:1167-1174). An HPLC apparatus of the HP1100 Series (Hewlett-Packard,Waldbronn, Germany) with attached fluorescence detector (G1321A) wasused; the operation of the systems and the evaluation of the data wascarried out with a HP-Chem-Station (Hewlett-Packard). 1 μL of the aminoacid solution to be analysed was mixed in an automatic preliminarycolumn derivatisation step with 20 μL ofo-phthalaldehyde/2-mercaptoethanol reagent (Pierce Europe BV,Oud-Beijerland, Netherlands).

The resultant fluorescing, thio-substituted isoindoles (Jones et al.,Journal of Chromatography (1983) 266: 471-482) were separated in acombined preliminary column (40×4 mm Hypersil ODS 5) and main column(Hypersil ODS 5, both columns obtained from CS-Chromatographie ServiceGmbH, Langerwehe, Germany) using a gradient program with an increasinglynon-polar phase (methanol). The polar eluent was sodium acetate (0.1molar, pH 7.2); the flow rate was 0.8 mL per minute. The fluorescencedetection of the derivatised amino acids was carried out at anexcitation wavelength of 230 nm and an emission wavelength of 450 nm.The amino acid concentrations were calculated by comparison with anexternal standard and asparagine as additional internal standard.

The results are shown in Table 2.

TABLE 2 L-Threonine (g/L) Strain 0 Hrs. 24 Hrs. 48 Hrs. 72 Hrs. DM368-2pZ1 0 0.46 1.27 1.50 DM368-2 pZ1thrE 0 0.68 1.71 2.04

The results are accompanied by the following figures:

FIG. 1: Map of the plasmid pCGL0040 containing the transposon Tn5531.The transposon is characterised as a non-hatched arrow.

FIG. 2: Map of the plasmid pZ1thrE containing the thrE gene.

Length data should be regarded as approximate. The abbreviations andsymbols employed have the following meaning:

-   -   BglII: restriction endonuclease from Bacillus globigii    -   EcoRI: restriction endonuclease from Escherichia coli    -   EcoRV: restriction endonuclease from Escherichia coli    -   SacI: restriction endonuclease from Streptomyces achromogenes    -   XbaI: restriction endonuclease from Xanthomonas badrii    -   XhoI: restriction endonuclease from Xanthomonas holcicola    -   Amp: ampicillin resistance gene    -   Kan: kanamycin resistance gene    -   ‘amp: 3’ part of the ampicillin resistance gene    -   oriBR322: replication region of the plasmid pBR322

1. A process for the preparation of L-threonine comprising fermentingL-threonine producing Corynebacterium or Brevibacterium in which theCorynebacterium glutamicum thrE gene encoding a threonine export proteinis overexpressed by increasing the copy number of said gene, andisolating said L-threonine produced by said Corynebacterium.
 2. Theprocess of claim 1 wherein the Corynbacterium glutamicum pye geneencoding pyruvate carboxylase is also overexpressed in saidCorynebacterium or Brevibacterium by increasing the copy number of saidgene.
 3. The process of claim 1, wherein the Corynebacterium glutamicumhom gene encoding for homoserine dehydrogenase is also overexpressed insaid Corynebacterium or Brevibacterium by increasing the copy number ofsaid gene.
 4. The process of claim 1, wherein the Corynebacteriumglutamicum hom^(dr) allele encoding a feedback-resistant homoserinedehydrogenase is also overexpressed in said Corynebacterium orBrevibacreriwn by increasing the copy number of said allele.
 5. Theprocess of claim 1, wherein Corynebacterium glutamicum mqo gene encodingmalate:quinone oxidoreductase is also overexpressed in saidCorynebacterium or Brevibacterium by increasing the copy number of saidgene.
 6. The process of claim 1, wherein Corynebacterium of the speciesCorynebacterium glutamicum are used.
 7. The process of claim 1, whereinBrevibacterium of the species Brevibacterium flavum are used.
 8. Aprocess for the preparation of L-threonine comprising fermentingL-threonine producing coryneform bacteria in which the Corynebacteriumglutamicum thrE gene encoding a threonine export carrier protein isoverexpressed by increasing the copy number of the thrE gene, andisolating said L-threonine produced by said coryneform bacteria, whereinsaid coryneform bacteria have been transformed with a plasmid vectorcomprising the C. glutamicum thrE gene encoding said threonine exportcarrier protein and said plasmid vector is pZ1thrE, which is depositedin Brevibacterium flavum under deposit number DSM12840.
 9. The processof claim 8, wherein Corynebacterium glutamicum pyc gene encodingpyruvate carboxylase is also overexpressed in said coryneform bacteriaby increasing the copy number of said gene.
 10. The process of claim 8,wherein the Corynebacterium glutamicum hom gene encoding for homoserinedehydrogenase is also overexpressed in said coryneform bacteriaincreasing the copy number of said gene.
 11. The process of claim 8,wherein the Corynebacterium glutamicum hom^(dr) allele encoding afeedback-resistant homoserine dehydrogenase is also overexpressed insaid Corynebacterium or Brevibacterium by increasing the copy number ofsaid allele.
 12. The process of claim 8, wherein the Corynebacteriumglutamicum mqo gene encoding malate:quinone oxidoreductase is alsooverexpressed in said coryneform bacteria by increasing the copy numberof said gene.
 13. The process of claim 8, wherein the coryneformbacteria of the genus Corynebacterium are used.
 14. The process of claim13, wherein the Corynebacterium of the species Corynebacteriumglutamicum are used.
 15. The process of claim 8, wherein the coryneformbacteria of the genus Brevibacterium are used.
 16. The process of claim15, wherein the Brevibacterium of the species Brevibacterium flavum areused.
 17. A process for the fermentative preparation of L-threoninecomprising: (a) fermenting L-threonine producing Corynebacterium orBrevibacterium bacteria in which a C. glutamicum thrE gene encoding athreonine export carrier protein is overexpressed by increasing the copynumber of said gene; and, wherein one or more of the coryneform genesselected from the group consisting of: the Corynebacrterium glutamicumpyc gene encoding pyruvate carboxylase, the Corynebacterium glutamicumhom gene encoding for homoserine dehydrogenase, the Corynebacteriumglutamicum hom^(dr) allele encoding a feedback-resistant homoserinedehydrogenase, and the Corynebacterium glutamicum mqo gene encoding formalate:quinone oxidoreductase are overexpressed by increasing the copynumber of said genes; (b) concentrating the L-threonine in thefermentation medium or in said coryneform bacteria; and (c) isolatingL-threonine from the fermentation medium or coryneform bacteria of step(b).
 18. A process for the fermentative preparation of L-threoninecomprising: (a) fermenting L-threonine producing coryneform bacteria inwhich a thrE gene encoding a threonine export carrier protein isoverexpressed by increasing the copy number of the gene; and, whereinone or more of the coryneform genes selected from the group consistingof: the Corynebacterium glutamicum pyc gene encoding pyruvatecarboxylase, the Corynebacterium glutamicum hom gene encoding forhomoserine dehydrogenase, the Corynebacterium glutamicum hom^(dr) alleleencoding a feedback-resistant homoserine dehydrogenase, and theCorynebacterium glutamicum mqo gene encoding for malate:quinoneoxidoreductase are overexpressed by increasing the copy number of saidgenes; (b) concentrating the L-threonine in the fermentation medium orin said coryneform bacteria; and (c) isolating L-threonine from thefermentation medium or coryneform bacteria of step (b) wherein saidcoryneform bacteria have been transformed with a plasmid vectorcomprising the C. glutamicum thrE gene encoding said threonine exportcarrier protein and said plasmid vector is pZ1thrE, which is depositedin Brevibacterium flavum under deposit number DSM12840.
 19. The processof claim 1, wherein said thrE gene comprises a polynucleotide encoding aprotein comprising the amino acid sequence of SEQ ID NO:
 2. 20. Theprocess of claim 19, wherein said polynucleotide comprises nucleotides398 to 1864 of SEQ ID NO:
 1. 21. The process of claim 20, wherein saidthrE gene comprises SEQ ID NO: 1 or SEQ ID NO:
 3. 22. A process for thepreparation of L-threonine comprising fermenting L-threonine producingCorynebacterium or Brevibacterium bacteria in which the Corynebacteriumglutamicum thrE gene encoding a threonine export protein isoverexpressed by operatively linking said gene to a promoter, andisolating said L-threonine produced by said Corynebacterium.
 23. Theprocess of claim 22, wherein the Corynebacterium glutamicum pyc geneencoding pyruvate carboxylase is also overexpressed in saidCorynebacterium or Brevibacterium by operatively linking said gene to apromoter.
 24. The process of claim 22, wherein the Corynebacteriumglutamicum hom gene encoding for homoserine dehydrogenase is alsooverexpressed in said Corynebacterium or Brevibacterium by operativelylinking said gene to a promoter.
 25. The process of claim 22, whereinthe Corynebacterium glutamicum hom^(dr) allele encoding afeedback-resistant homoserine dehydrogenase is also overexpressed insaid Corynebacterium or by Brevibacterium operatively linking saidallele to a promoter.
 26. The process of claim 22, wherein theCorynebacterium glutamicum mqo gene encoding malate:quinoneoxidoreductase is also overexpressed in said Corynebacterium orBrevibacterium by operatively linking said gene to a promoter.
 27. Theprocess of claim 22, wherein Corynebacterium bacteria of the speciesCorynebacterium glutamicum are used.
 28. The process of claim 22,wherein the Brevibacterium bacteria of the species Brevibacterium flavumare used.
 29. A process for the preparation of L-threonine comprisingfermenting L-threonine producing coryneform bacteria in which theCorynebacterium glutamicum thrE gene encoding a threonine export carrierprotein is overexpressed by operatively linking said thrE gene to apromoter, and isolating said L-threonine produced by said coryneformbacteria, wherein said coryneform bacteria have been transformed with aplasmid vector comprising the C. glutamicum thrE gene encoding saidthreonine export carrier protein and said plasmid vector is pZ1thrE,which is deposited in Brevibacterium flavum under deposit numberDSM12840.
 30. The process of claim 29, wherein the Corynebacteriumglutamicum pyc gene encoding pyruvate carboxylase is also overexpressedin said coryneform bacteria by operatively linking said gene to apromoter.
 31. The process of claim 29, wherein the Corynebacteriumglutamicum hom gene encoding for homoserine dehydrogenase is alsooverexpressed in said coryneform bacteria by operatively linking saidgene to a promoter.
 32. The process of claim 29, wherein said coryneformbacteria also overexpress by operatively linking the Corynebacteriumglutamicum hom^(dr) allele encoding a feedback-resistant homoserinedehydrogenase to a promoter.
 33. The process of claim 29, wherein theCorynebacterium glutamicum mqo gene encoding malate:quinoneoxidoreductase is also overexpressed in said coryneform bacteria byoperatively linking said gene to a promoter.
 34. The process of claim29, wherein coryneform bacteria of the genus Corynebacterium are used.35. The process of claim 34, wherein Corynebacterium of the speciesCorynebacterium glutamicum are used.
 36. The process of claim 29,wherein coryneform bacteria of the genus Brevibacterium are used. 37.The process of claim 36, wherein Brevibacterium of the speciesBrevibacterium flavum are used.
 38. A process for the fermentativepreparation of L-threonine comprising: (a) fermenting L-threonineproducing Corynebacterium or Brevibacterium bacteria in which a C.glutamicum thrE gene encoding a threonine export carrier protein isoverexpressed by operatively linking said gene to a promoter; and,wherein one or more of the coryneform genes selected from the groupconsisting of: the Corynebacterium glutamicum pyc gene encoding pyruvatecarboxylase, the Corynebacterium glutamicum hom gene encoding forhomoserine dehydrogenase, the Corynebacterium glutamicum hom^(dr) alleleencoding a feedback-resistant homoserine dehydrogenase, and theCorynebacterium glutamicum mqo gene encoding for malate:quinoneoxidoreductase are overexpressed by operatively linking said genes to apromoter; (b) concentrating the L-threonine in the fermentation mediumor in said coryneform bacteria; and (c) isolating L-threonine from thefermentation medium or coryneform bacteria of step (b).
 39. A processfor the fermentative preparation of L-threonine comprising: (a)fermenting L-threonine producing coryneform bacteria in which a thrEgene encoding a threonine export carrier protein is overexpressed byoperatively linking said gene to a promoter; and, wherein one or more ofthe coryneform genes selected from the group consisting of: theCorynebacterium glutamicum pyc gene encoding pyruvate carboxylase, theCorynebacterium glutamicum hom gene encoding for homoserinedehydrogenase, the Corynebacterium glutamicum hom^(dr) allele encoding afeedback-resistant homoserine dehydrogenase, and the Corynebacteriumglutamicum mqo gene encoding for malate:quinone oxidoreductase areoverexpressed by operatively linking said genes to a promoter; (b)concentrating the L-threonine in the fermentation medium or in saidcoryneform bacteria; and (c) isolating L-threonine from the fermentationmedium or coryneform bacteria of step (b) wherein said coryneformbacteria have been transformed with a plasmid vector comprising the C.glutamicum thrE gene encoding said threonine export carrier protein andsaid plasmid vector is pZ1thrE, which is deposited in Brevibacteriumflavum under deposit number DSM12840.
 40. The process of claim 22,wherein said thrE gene comprises a polynucleotide encoding a proteincomprising the amino acid sequence of SEQ ID NO:
 2. 41. The process ofclaim 40, wherein said polynucleotide comprises nucleotides 398 to 1864of SEQ ID NO:
 1. 42. The process of claim 41, wherein said thrE genecomprises SEQ ID NO: 1 or SEQ ID NO: 3.